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Sample GSM2609106

Query DataSets for GSM2609106

Status

Public on Apr 25, 2018

Title

input for "SON TSA-Seq (Ab2) Condition 2 (TSA+Sucrose)"

Sample type

SRA

Source name

bone marrow

Organism

Homo sapiens

Characteristics

cell line: K562
disease: chronic myelogenous leukemia (CML)
tsa-seq target: SON

Treatment protocol

TSA labeling was done in independent cell batches. For each batch, cells were divided equally and stained according to four labeling protocols: “Condition 1”, “Condition 2”, “Condition 3”, and “No 1o control”. Identical staining conditions were used except that 50% sucrose was included in the TSA staining buffer for the “Condition 2” protocol, 0.75mM DTT in addition to 50% sucrose was added in the TSA staining buffer for the “Condition 3” protocol, and the anti-SON primary antibody was omitted for the “No 1o control” protocol. Cells were fixed by adding 10 ml of 8% paraformaldehyde (Sigma-Aldrich) freshly prepared in CMF-PBS (calcium, magnesium-free phosphate buffered saline) to 40 ml of cells in culture media, incubating for 20 min at room temperature (RT), and quenching aldehydes by addition of 5.6 ml of 1.25 M glycine (Fisher Scientific) and incubation for 5 min at RT. All incubation steps used a rotating platform to mix the cell suspension. Cells were then pelleted by centrifugation and resuspended in CMF-PBS buffer with 0.5% Triton-X100 (Sigma-Aldrich) for 30 min at RT. All centrifugations were for 5 min at 100-200g. Cells were centrifuged at RT and resuspended in 1.5% H2O2 (Fisher Scientific) in CMF-PBS for 1h at RT to quench endogenous peroxidases, followed by 3 washes at RT in PBST (0.1%Triton-X100/CMF-PBS) by repeated centrifugation and cell pellet resuspension. Cells were resuspended in IgG Blocking Buffer (5% Normal Goat Serum (Sigma-Aldrich) in PBST) at a concentration of 10^7 cells/ml for 1 hr, followed by centrifugation and resuspension in primary antibody solution (Rabbit-anti-SON primary antibodies (Ab1, Atlas antibodies cat # HPA023535) diluted 1:1000 or 1:2000 in IgG Blocking buffer, or rabbit polyclonal-anti-SON primary antibodies (Ab2) diluted 1:2000 in IgG Blocking buffer, or mouse-anti-phosphorylated SC35 primary antibodies (Sigma-Aldrich cat # S4045) diluted 1:500 in IgG Blocking buffer, or mouse monoclonal anti-lamin A/C primary antibody (Clone 5G4) diluted 1:1000 in IgG Blocking buffer, or mouse monoclonal anti-lamin B primary antibodies (Clone 2D8) diluted 1:1000 in IgG Blocking buffer) at 10^7 cells/ml, or mouse monoclonal anti-RNA polymerase II primary antibodies (clone 4H8) (Millipore cat # 05-623) at 0.5 x 107 cells/ml and incubated 20-24 hrs at 4°C (See METHODS for more information about anti-lamin antibodies). For the no primary control, cells were incubated in IgG Blocking buffer only. Cells were then washed 3x with PBST, resuspended in IgG Blocking buffer with secondary antibodies at 10^7 cells/ml, and incubated 20-24 hrs at 4°C. Secondary antibodies (Jackson, ImmunoResearch) used were HRP conjugated, goat-anti-rabbit secondary antibody diluted 1:1000 or HRP conjugated goat-anti-mouse secondary antibody diluted 1:200 or 1:1000. Cells then were washed 3x in PBST and resuspended in reaction buffer A (CMF-PBS for Condition 1, or CMF-PBS with 50% sucrose for Condition 2, or CMF-PBS with 50% sucrose and 1.5mM DTT for Condition 3) at 2 x 10^7 cells/ml. Equal volumes of buffer B (8mM tyramide-biotin stock solution diluted 1:5000 with 0.003% H2O2 in CMF-PBS for Condition 1, or CMF-PBS with 50% sucrose for Conditions 2 and 3) were then added. Cells were incubated for 10 min and then washed 3x in PBST at RT. Validation of TSA labeling is done by biotin immunostaining.

Growth protocol

K562 Cells were grown according to the ENCODE protocol, in RPMI with 10% FBS (Sigma-Aldrich) and Antibiotic-antimycotic (GIBCO) at density of 0.4 x 10^6-0.75 x 10^6 /ml

Extracted molecule

genomic DNA

Extraction protocol

Cell lysis and protease digestion were done at 55°C with constant mixing in 10mM Tris, 10mM EDTA, 0.5% SDS and 500µg/ml proteinase K (NEB) for 8-12 hrs (10^7 cells/ml). NaCl was added for a final 0.2 M concentration and the lysate incubated 20-24 hrs at 65°C with constant mixing to reverse formaldehyde crosslinking. DNA was extracted 2x using an equal volume of phenol (Fisher)/chloroform/isoamyl alcohol (25:24:1) and 1x with an equal volume of chloroform/isoamyl alcohol (24:1). Tubes were kept at 55°C for 1h with the cap off to evaporate phenol residues, and then treated with 25 µg/ml RNaseA (Qiagen) at 37o C for 1hr and extracted again. DNA was then precipitated with 1/10 volume of 3M Na Acetate, pH 5.2, and 2 volumes of 100% EtOH at -20 oC for 20 min – 2 hrs.
For each of the TSA-Seq experiment staining conditions, input and Streptavidin pull-down DNA were used to make sequencing libraries, using the Kapa Hyper Prep Kit (Kapa Biosystems). DNA was blunt-ended, 3’-end A-tailed, and ligated to indexed adaptors with 6nt barcodes. PCR amplification selectively enriched for those fragments with adapters on both ends. Amplification was carried out for 8-15 cycles with the Kapa HiFi polymerase (Kapa Biosystems, Woburn, MA). An Agilent bioanalyzer DNA high-sensitivity chip (Agilent, Santa Clara, CA) was used to determine library fragment sizes. Library fragment concentrations were measured by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA) prior to pooling and sequencing.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

SON_Ab2_TSA-score_signal_TSA+Sucrose.bw

Data processing

Library strategy: TSA-Seq
The raw .bcl files were converted into demultiplexed compressed fastq files using the bcl2fastq v1.8.2 and later v2.17.1.14 conversion software (Illumina).
Raw sequencing reads were first mapped to the human genome (UCSC hg19) using Bowtie 2 version 2.0.2 with default parameters. Chromosome Y was excluded in the reference genome to reduce mapping bias since the K562 cell line was derived from a female.
PCR duplicates reads were removed using samtools version 0.1.19 with default parameters.
We normalized and processed the TSA-Seq data using a sliding window approach as follows. The normalized SON-TSA score was calculated as the log 2 ratio of (TSA-Seq pulldown reads in a 20 kb window divided by total mapped reads for this pulldown sample) / (input reads in the same 20 kb window divided by total mapped reads for this input). This approach can be interpreted as the fold change between the pulldown sample and the input corrected by the total number of mapped reads in each sample. The window slides on the genome with a step size of 100 bp.
We partitioned the genome into non-overlapping 20 kb bins, averaging the TSA-Seq score over the 100 bp intervals within this bin for the new TSA-Seq 20 kb score. The values for these 20 kb bins were then smoothed by convolution using a Hanning window of length 21 (21 x 20 kb or 420 kb). This new smoothed TSA-Seq score with 20 kb sampling was used for SON TSA-Seq decile calculations and comparisons with other genome features
Genome_build: hg19
Supplementary_files_format_and_content: normalized TSA signals were in bigWig format, and the 10 deciles are in BED format. Other data files from analyzing public datasets are in bigWig or bigBed formats.

Submission date

May 08, 2017

Last update date

May 15, 2019

Contact name

Andrew S Belmont

E-mail(s)

asbel@illinois.edu

Phone

(217) 244-2311

Organization name

University of Illinois at Urbana-Champaign